GlnB and its homologs are members of the PII protein family and are central metabolic regulators in almost all prokaryotic cells, including all major pathogens. It is generally agreed that GlnB-like proteins sense the carbon-nitrogen balance in the cell and respond by assuming a mixture of two forms: one form signals nitrogen excess and the other signals nitrogen limitation. These two forms then affect a variety of cellular process by direct protein-protein interactions with a number of receptor proteins in the cell. Finally, a number of structures of the nitrogen-excess form have been solved. Despite this knowledge, some very central issues remain unresolved and are the focus of this proposal. First, it is clear that GlnB-like proteins bind ATP, but this ATP-binding has not been thought to be of physiological importance. However, strong preliminary evidence suggests that changes in ATP levels affect GlnB function in the cell, which implies that GlnB integrates carbon, nitrogen and energy signals. Examining the role of energy status on GlnB function is the first aim of this proposal. The focus of Aim II is to better understand the currently unknown structure of the nitrogen-limitation form of GlnB. Preliminary evidence suggests that it differs in dramatic ways from the known structures of the nitrogen-excess form. Direct structural analysis by NMR and X-ray crystallography will be complemented by the existence of conformationally altered GlnB variants. This aim also directly tests the recent model of the binding site of 1-ketoglutarate, which is necessary for achieving the form signaling nitrogen limitation, by a combination of genetic and biochemical approaches. Finally, Aim III addresses the interaction of GlnB with AmtB, an integral membrane protein that also serves as an ammonia gas channel. An important result of this interaction is to directly affect GlnB levels in the cell and therefore GlnB's ability to interact with other proteins. A combination of biochemical physiological assays will determine the nature and importance of the regulation of this GlnB-AmtB interaction. All these aims have a very high likelihood of success because the necessary protein variants are already in hand and the biochemical and genetic tools are available. The result of the proposed experiments will be a dramatically improved description of how GlnB homologs in all organisms integrate signals of nitrogen, carbon and energy status at the molecular level. This project addresses the intersection of carbon, nitrogen and energy sensing by the PII protein system found in almost all prokaryotes. Because of its centrality, it directly or indirectly affects both beneficial (antibiotic production) and harmful (toxin production) microbial processes. The results will therefore help explain how both beneficial and pathogenic organisms sense and respond to their environment. [unreadable] [unreadable] [unreadable]